Cancer has been postulated to be a disorder of differentiation. We propose to address the relationship of altered differentiation to multistage carcinogenesis by means of an in vitro cellular transformation system using mouse epidermal cells. Mouse epidermis is readily transplantable in vivo and in vitro and is the system for classical as well as recent developments in the mechanisms of tumor promotion and malignant conversion. Furthermore, epidermal cells exhibit 4 stages of differentiation similar to human epidermal cells, with well characterized morphologic and immunologic markers. Three hypotheses are to be tested: First, that tumor promoters act to permit the clonal expansion of initiated cells defective in sensitivity to differentiation signals, by inducing terminal differentiation in normal cells competent in sensitivty to such signals. To test this, the potency of certain phorbol ester and non-phorbol ester tumor promoters in inducing the differentiation of epidermal clone 271c cells (derived in our laboratory on the basis of normal response to Ca++ as a differentiation signal) will be compared to their effectiveness as tumor promoters as reported in vivo, and measured in vitro using 271c cells in an assay recently reported by us. Secondly, that tumor promoters enhanced benign tumor promotion but not the malignant conversion of epidermal cells. "Initiated" subclones of 271c cells will be used as targets for certain tumor promoting and mutagenic agent, alone and in mixed culture with normal cells, and after transplantation in vivo to denuded skin sites. Enhancement of transformation will be evaluated based on growth in soft agar and formation of benign or malignant tumors in vivo. Thirdly, that anti-promoting agents sensitize initiated cells to differentiation signals. Capacity of "initiated" 271c cells to respond to differentiation signals in the presence of retinoids will be examined. The conditions determining the enhancement vs inhibitory effects of retinoic acid on transformation will be approached by studies of the influence of target cell cycle stage and structural modifications of retinoid analogs. The mechanisms of carcinogenesis proposed herein for study at the biologic level will be used as a basis for future studies of epithelial cell transformation at the molecular level. These studies are important for assessing the potential of differentiation-inducing agents in chemoprevention or treatment of human cancer.